We wish to understand how the binding of acetylcholine and other agonists by the nicotinic cholinergic receptor controls the permeability of the post-synaptic membrane and to understand how drugs and toxins interact with that receptor when they block the action of acetylcholine. Torpedo electric tissue will be fractionated to isolate post-synaptic membranes that will be used in studies of receptor structure and function. Radioligand and fluorescence binding assays will be used to define the kinetic and equilibrium binding constants characterizing receptor occupancy by agonists and antagonists. The permeability response will be measured by the flux of 22Na+ and other isotopes from the membranes. We will study the effects on the response of known concentrations of agonists and antagonists when present for times as short as 10 msec. The results of these functional studies will be used to determine how agonists binding results in the opening of the ion channel and then desensitization. We focus on the actions of amine non-competitive antagonists, including many local anesthetics, that block the action of acetylcholine by binding to a distinct "anesthetic" site on the receptor. We will identify the location of the binding sites for agonists and non-competitive antagonists within the structure of the receptor. Binding sites will be labeled covalently by appropriate radiolabeled affinity reagents, and the receptor will be degraded to identify the labeled peptides in terms of the primary amino acid sequence of the receptor subunits. To determine how the ligand binding sites are oriented within the three-dimensional structure of the receptor, we will use biochemical and immunological labeling procedures to identify receptor domains that are exposed at the extracellular surface, within the lipid bilayer, or on the cytoplasmic surface. We will also determine the linear distances between the ligand binding sites, the membrane bilayer and the cytoplasmic surface of the receptor from the energy transfer observed between appropriate fluorescent labels.